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Discovery of GSK2656157: An Optimized PERK Inhibitor Selected for Preclinical Development Jeffrey M Axten, Stuart P Romeril, Arthur Shu, Jeffrey M Ralph, Jesus R. Medina, Yanhong Feng, William Hoi Hong Li, Seth W Grant, Dirk A. Heerding, Elisabeth Minthorn, Thomas Mencken, Nathan Gaul, Aaron Goetz, Thomas B. Stanley, Annie M Hassell, Robert T Gampe, Charity Atkins, and Rakesh Kumat ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/ml400228e • Publication Date (Web): 12 Aug 2013 Downloaded from http://pubs.acs.org on August 23, 2013
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ACS Medicinal Chemistry Letters
Discovery of GSK2656157: An Optimized PERK Inhibitor Selected for Preclinical Development Jeffrey M. Axten,*,† Stuart P. Romeril,† Arthur Shu,† Jeffrey Ralph,† Jesús R. Medina,† Yanhong Feng,† William Hoi Hong Li,† Seth W. Grant,† Dirk A. Heerding,† Elisabeth Minthorn,† Thomas Mencken,† Nathan Gaul, ‡ Aaron Goetz,§ Thomas Stanley,§ Annie M. Hassell,║ Robert T. Gampe,║ Charity Atkins† and Rakesh Kumar† GlaxoSmithKline Research and Development, †
Oncology Research, Protein Dynamics DPU, Collegeville, PA 19426, United States
‡
Screening and Compound Profiling, Collegeville, PA 19426, United States
§
Screening and Compound Profiling, Research Triangle Park, NC 27713, United States
║
Biomolecular Structure, Computational and Structural Chemistry, Research Triangle Park, NC 27709, United States
KEYWORDS: PERK, UPR, kinase, lead optimization, structure-activity relationship, fluorine interaction ABSTRACT: We recently reported the discovery of GSK2606414 (1), a selective first in class inhibitor of protein kinase R (PKR)like endoplasmic reticulum kinase (PERK) which inhibited PERK activation in cells and demonstrated tumor growth inhibition in a human tumor xenograft in mice. In continuation of our drug discovery program, we applied a strategy to decrease inhibitor lipophilicity as a means to improve physical properties and pharmacokinetics. This report describes our medicinal chemistry optimization culminating in the discovery of the PERK inhibitor GSK2656157 (6), which was selected for advancement to preclinical development.
Increased endoplasmic reticulum (ER) stress resulting from nutrient deprivation and unfolded protein accumulation is associated with debilitating conditions such as neurodegeneration, heart disease, diabetes, and cancer.1 To maintain ER homeostasis, the unfolded folded protein response (UPR) coordinates an adaptive cellular signaling cascade to alleviate the impact of the stress and enhance cell survival.2 Protein kinase R (PKR)-like ER kinase (PERK) is one of three primary effectors of the UPR.3-4 Once activated, PERK phosphorylates eukaryotic initiation factor 2α (eIF2α) at serine 51 which inhibits the ribosome translation initiation complex and reduces overall protein synthesis.5-7 The reduction in translation reduces the ER burden, providing time for the cell to process or degrade the accumulated unfolded proteins. Although global protein synthesis is decreased, there is also specific increased transcription of certain messages regulated by downstream PERK effectors, such as ATF4, which activate genes that enhance UPR function. For example, in situations of extreme hypoxia or nutrient starvation, UPR activation is associated with increased vascularization. A number of studies using genetic manipulation or siRNA knock down provide evidence that PERK function contributes to transcriptional activation and up-regulation of pro-angiogenic genes.8-14 Therefore, inhibiting PERK in cancer cells may limit their ability to thrive under hypoxia or nutrient deprived conditions and lead to apoptosis or tumor growth inhibition. Recently we reported the discovery and characterization of GSK2606414 (1), a highly selective, first in class inhibitor of PERK that demonstrated tumor growth inhibition in a human tumor xenograft in mice.15 In this communication we disclose the medicinal chemistry optimization leading to the discovery of the preclinical development candidate GSK2656157 (6), which was recently reported with extensive biological characterization.16
Although compound 1 served as an excellent, orally available tool to elucidate the function of PERK in cells and animals, we sought improvements to the physicochemical properties, metabolism and pharmacokinetics. Data from in vitro metabolic studies showed that 1 broadly inhibited cytochrome P450s in human liver microsomes, with sub-micromolar inhibition of CYP2C8 (IC50 = 0.89 µM, Table 2). We suspected that the broad inhibitory activity against cytochrome P450s was related to the overall lipophilicity of the molecules and electronic nature of the arylacetamide based on our previous work relating this part of the molecule to in vivo rat clearance.15 Thus, our optimization strategy to increase polarity focused on modifying the aryl acetamide. We specifically targeted heteroaryl acetamides to minimize molecular weight gain and to evenly distribute polarity throughout the molecules, with an emphasis on the design of analogs with cLogP values 25
>25
>25
19.7
>25
8
2.9
>25
19.2
>25
23.7
9.09
7.4
5.83
12
2.5
>25
>25
>25
>25
>25
20.5
>25
CYP450s with molecular probe listed under specific isoform. For assay details, see supporting information. Calculated using BioByte cLogP (the calculated logarithm of the 1‑octanol–water partition coefficient of the non-ionized molecule, see www.biobyte.com.cdextromethorphan was used as the probe in this experiment. a
b
Table 3. Kinase inhibiton summary for compounds 6, 8, and 12. EIF2AK1 HRI IC50 (nM)a
HRI/PERK IC50 ratio
EIF2AK2 (PKR) IC50 (nM) a
PKR/PERK IC50 ratio
EIF2AK4 (GCN2) IC50 (nM) b
GCN2/PERK IC50 ratio
X/300 Kinases Inhibited >80% @ 10 µMc
6
460
511
905
1,006
3,388
3,764
17
8
37
74
359
718
776
1,552
39
12
61
23
99
37
ND
ND
ND
Compound
Data from a single 10-point dose response performed by Reaction Biology Corp. (http://www.reactionbiology.com). bValues reported are the average of at least two experiments (see Experimental Section for details). cSee supporting information for complete kinase profile. a
three analogs since it potently inhibits both HRI and PKR (IC50s = 37 and 99 nM, respectively) with < 50-fold selectivity for PERK. Compounds 6 and 8, the two most selective PERK inhibitors within the EIF2AK family were profiled against a panel of 300 kinases. In line with the EIF2AK data, 6 continued to demonstrate superior kinase selectivity, inhibiting only 17/300 kinases >80% at 10 µM, whereas compound 8 inhibited 39/300 kinases >80% at the same concentration. Pharmacokinetic studies were performed in mouse, rat and dog for compound 6 (Table 4). Data collected from i.v./p.o. crossover studies showed that compound 6 was well absorbed providing good exposure, high oral availability, and low to moderate blood clearance in mouse, rat and dog. Estimated volume of distribution at steady-state was low in rodents and moderate to high in the dog. Half-lives were less than 2 hours in the mouse and rat (T1/2 = 1.25 and 1.4 h, respectively), which influenced the use of twice a day dosing in efficacy studies in mice. We recently reported the results of extensive biological evaluation of 6 in cell culture and in vivo.16 Chemical stress induced PERK activation was inhibited by 6 in multiple cell lines, with corresponding decreases in eIF2α phosphorylation and downstream transcriptional activation. In efficacy studies, oral treatment with 6 resulted in dose-dependent inhibition of multiple human tumor xenografts growth in mice. The tumor growth inhibition was mechanistically associated with an anti-angiogenic affect, which is in agreement with observations reported using genetic means to down-regulate PERK in mouse tumor studies.8-10 In addition, after
treatment with 6 for several weeks we observed a pancreas specific phenotype in mice characteristic of genetic PERK ablation.20-22 Taken together, the data strongly supported that the observed pharmacologic effects upon treatment with 6 were associated with PERK inhibition. The collective biological effects, exquisite kinase selectivity, and pharmacokinetic profile supported the selection of 6 (GSK2656157) for progression to preclinical development.
Table 4. Pharmacokinetic parameters of compound 6.a Mousec
Ratd
Doge
i.v. dose (mg/kg)
2.0
2.2
2.9
AUC(0-inf) ng*h/mL
3270 (2817-4085)
3921.0 (3226.84615.2)
3128.5 (2577.13594.8)
CLb (mL/min/kg)
10.5 (8.2-11.8)
9.5 (8.0-10.9)
15.5 (13.418.2)
Vdss (L/kg)
0.72 (0.50-0.86)
0.6 (0.6)
2.8 (2.8-2.9)
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ACS Medicinal Chemistry Letters T½ (h)
1.25 (1.13-1.36)
1.4 (1.3-1.5)
3.1 (2.7-3.6)
oral dose (mg/kg)
13.4
4.3
5.2
AUC (0-inf) ng*h/mL
13378.6 (13110.613645.5)
8015.7 (7174.58856.8)
6210.0 (5274.97144.2)
Oral F(%)
52b
~100
~100
Data is reported as a mean, with ranges provided in parentheses. bBioavailability (F%) was estimated using mean AUC (0-t) values due to the non-crossover study design. cMouse i.v. (bolus, n=3) 1% DMSO and 20% Captisol in saline, pH = 4 ; p.o. (suspension, n = 2): 2% DMSO and 40% PEG 400 in water, pH = 4.0. d Rat i.v. (60 minute infusion, n = 2): 1%DMSO and 20% Captisol in saline, pH = 4; oral (solution, n = 2): 1% DMSO and 20% PEG 400 in water, pH=4. eDog i.v. (60 minute infusion, n = 3): 1% DMSO and 20% Captisol in saline, pH = 6.5; p.o. (solution, n = 3) 1%DMSO and 40% PEG 400 in water, pH = 4.9. a
In summary, our PERK inhibitor lead optimization was guided by a strategy to decrease analog lipophilicity while maintaining the potency and exquisite kinase selectivity of tool inhibitor 1. We focused on heteroaryl acetamide analogs to minimize molecular weight gain and designed a series of inhibitors with low to very low in vivo rat blood clearance. Fluorination of the indoline 4postion was important for the recovery of potent biochemical and cell potency, and the optimized 4-fluorindoline analogs 6, 8, and 12 had favorable pharmacokinetics and lower levels of P450 inhibition in human liver microsomes. Expanded profiling established the superior kinase selectivity of 6 which was selected as a preclinical development candidate.
ASSOCIATED CONTENT Supporting Information Available: General Synthetic scheme and experimental procedures for the synthesis of compounds 2-12, DMPK and biological assay descriptions, crystallographic methods, and kinase selectivity profile information. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION Corresponding Author * (J.M.A.) Phone: 610-270-6368. E-mail:
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a novel PERK kinase inhibitor with anti-tumor and anti-angiogenic activity. Cancer Res. 2013, 73, 1993-2002. 17. Shah, P.; Westwell, A.D. The role of fluorine in medicinal chemistry. J. Enzyme Inhib. Med. Chem. 2007, 22, 527-540. 18. Müller, K.; Faeh, C.; Diederich, F. Fluorine in Pharmaceuticals: Looking Beyond Intuition. Science 2007, 317, 1881-1886. 19. Zhou, P.; Zou, J.; Tian, F.; Shang, Z. Fluorine Bonding – How Does It Work In Protein-Ligand Interactions? J. Chem. Inf. Model. 2009, 49, 2344-2355. 20. Harding, H.P.; Zeng, H.; Zhang, Y.; Jungries, R.; Chung, P.; Plesken, H.; Sabatini, D.D.; Ron, D. Diabetes Mellitus and Exocrine Pancreatic Dysfunction in Perk-/- Mice Reveals a Role for Translational Control in Secretory Cell Survival. Mol. Cell 2001, 7, 1153–1163. 21. Zhang, P.; McGrath, B.; Li, S.; Frank, A.; Zambito, F.; Reinert, J.; Gannon, M.; Ma, K.; McNaughton, K.; Cavener, D.R. Mol. Cell. Bio. 2002, 22, 3864-3874. 22. Gao, Y.; Sartori, D.J.; Li, C.; Yu, Q.; Kushner, J.A.; Simon, M.C.; Diehl, J.A. PERK Is Required in the Adult Pancreas and Is Essential for Maintenance of Glucose Homeostasis. Mol. Cell. Bio. 2012, 32, 5129-5139.
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